Dissection of the hypoxia induced transcription factor complex bound to the erythropoietin enhancer under hypoxic conditions has led to the identification of Hypoxia-inducible factor-1 (HIF-1). From here the whole cascade of cellular O₂ sensing was followed back ultimately leading to the elucidation of cellular O₂ sensors, the prolyl and asparagyl hydroxylases that regulate abundance and activity of oxygen-regulated -subunit of HIF-1. Assembly of the HIF- 1 complex then requires dimerization with constitutive nuclear b-subunit. Mobility studies of fluorescently labelled HIF-1 subunits by fluorescence recovery after photo bleaching (FRAP) revealed that HIF-1a migrates more slowly than HIF-1b within the nucleus indicating that both subunits do not immediately "find" each other but may be prone to modification prior to dimerization. To localize HIF-1 subunits within the nucleus of hypoxic cells we applied 2-photon-laser microscopy (2PLM). HIF-1 assembly was then studied in living cells in a specialized hypoxic chamber mounted on the microscopic stage which allows the in vivo analysis under a well defined oxygen tension. Assembly of the HIF-1 complex was analysed by fluorescence resonance energy transfer (FRET) and determined the distance of a- and b-subunits of the transcriptionally active HIF-1 complex bound to DNA. Interestingly, the N- and C-terminus of HIF- 1a and -b were closely co- localized. This organization of the HIF complex is different from so far described two dimensional models but is in line with the very recent structural consideration that heterodimer formation of the HIF complex relies on anti- parallel association of the PAS B domains within the a- and b-subunit.